Locally increased Dysferlin expression improves left-ventricular function by stabilizing membrane nanodomains of cardiomyocytes in the myocardial infarction border zone

Dysferlin, a transmembrane protein with multiple Ca²⁺-sensitive C2 domains, is found in the sarcolemma and vesicle membranes of striated muscle cells. Upon Ca²⁺ entry, Dysferlin is thought to facilitate vesicle fusion and membrane repair. Dysferlin loss-of-function mutations are associated with progressive muscular dystrophies and dilated cardiomyopathy. However, its role in protecting subcellular membrane domains in ventricular myocytes (VMs) after myocardial infarction (MI) remains unclear. Here, we hypothesize that Dysferlin prevents from cell death and loss-of-function in VMs within the MI border zone by stabilizing the transverse-axial tubule (TAT) network and the intercalated disc (ICD) cell-cell contact sites.

Results:
Using Stimulated Emission Depletion (STED) nanoscopy on immunolabeled VMs, punctate Dysferlin signals were localized in close proximity to the TAT network and the ICDs. Anti-Dysferlin co-immunoprecipitation experiments and subsequent data-independent acquisition mass spectrometry (DIA-MS) from WT and KO left-ventricular (LV) tissues identified the cardiac interactome of Dysferlin, identifying interactions with proteins of the TAT system and the gap junction hemichannel Connexin-43 (CX43). Importantly, 1 week after MI surgery induced by ligation of the left anterior descending (LAD) artery, investigator-blinded echocardiography showed that Dysferlin-KO mice exhibited larger infarct sizes and reduced LV ejection fraction compared to WT mice (mean±SEM: 21±2% vs. 28±2%, n=20/30 mice, P<0.05). In WT mice, myocardial Dysferlin protein expression increased by 148% compared to sham-operated mice post-MI. More specifically, in the MI border zone, which experiences a high ischemic and mechanical stress, local Dysferlin expression rose to 230% post-MI. Hence, we employed DIA-MS in a spatial proteomics approach to analyse the proteomic profile of the MI zone, border zone, and remote zone in WT vs. KO hearts 1 week post-MI (each n=5 mice). Overall, we consistently quantified 4.360 proteins across all LV samples, identifying local and genotype-specific proteotypes post-MI. Comparing WT vs. KO samples, DIA-MS revealed 725 differentially abundant proteins in the border zone pairwise comparison, underscoring the crucial role of Dysferlin in LV remodelling in MI. Interestingly, immunohistology showed VMs with significantly disrupted TAT networks and elongated ICD regions in the MI border zone. On the subcellular level, STED nanoscopy revealed Dysferlin clusters that highly decorated residual TAT structures as well as disorganized ICD membrane folds in the MI border zone. Detailed STED signal analysis confirmed the local accumulation of Dysferlin at residual TAT structures and fragmented CX43 plaques in the MI border zone.

Conclusion:
Our data point out a significant role for Dysferlin in VMs post-MI, while Dysferlin deficiency leads to larger infarct sizes and reduced LV systolic function. Subcellularly, Dysferlin stabilizes critical membrane nanodomains in VMs namely residual TAT structures for preserved Ca²⁺ signalling as well as ICD membrane folds for cell-cell communication of the MI border zone. Therefore, Dysferlin may emerge as a novel therapeutic target to control membrane integrity and to prevent from loss-of-function and cell death in VMs post-MI.